Synthesis of thymosin α1

ABSTRACT

Thymosin α 1 , was chemically synthesized by the fragment condensation of the protected amino terminal tetradecapeptide with the protected carboxyl terminal tetradecapeptide and by solid phase peptide synthesis. Thymosin α 1  is active as an agent which affects regulation, differentiation and function of thymus dependent lymphocytes (T cells).

RELATED APPLICATIONS

This application is a continuation-in-part of copending application Ser.No. 871,563, filed Jan. 23, 1978 which in turn is a continuation-in-partof Ser. No. 789,898 filed Apr. 22, 1977, now abandoned.

BACKGROUND OF THE INVENTION

Several polypeptide factors present in the thymus gland have beenimplicated to play important roles in the development and maintenance ofimmunological competence in man and in animals. The importance of theimmune system in the defense against cancer and tumor cells is nowwidely recognized. In recent years, a few polypeptides shown to be ableto stimulate maturation, differentiation and function of T cells havebeen isolated from bovine thymus. Among them, the acidic peptidethymosin α₁, has been intensively studied. Its structure and activityhave been described in U.S. Pat. No. 4,079,127. Additionally, it shouldbe noted that in the parent case thereof, U.S. patent application Ser.No. 736,638 filed Oct. 28, 1976, the structure of thymosin α₁, waserroneously given as that of [Asn² ]-thymosin α₁.

The correct structure of thymosin α₁ is as follows:

    Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH

description of the invention

Thymosin α₁ has been chemically synthesized in a preferred embodiment bythe condensation of the novel protected carboxyl terminaltetradecapeptide of the sequence

    TFA . H-Asp(OBzl)-Leu-Lys(Z)-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl                          I

with the novel protected amino terminal tetradecapeptide of the sequence

    Ac-Ser(Bzl)-Asp(OBzl)-Ala-Ala-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH                            II

followed by removal of the protecting groups.

The aforesaid coupling reaction can be carried out utilizing procedureswell known in solution phase peptide synthesis. Thus, for example, theamino terminal tetradecapeptide can be activated with a carbodiimidecoupling agent such as dicyclohexylcarbodiimide (DCC) and1-hydroxybenzotriazole (HOBT) and the ensuing active ester is reactedwith the trifluoroacetic acid (TFA) salt of the carboxyl terminaltetradecapeptide to yield the desired thymosin α₁ in protected form ofthe following structure:

    Ac-Ser(Bzl)-Asp(OBzl)-Ala-Ala-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-Asp(Bzl)-Leu-Lys(Z)-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl III

removal of the protecting groups from the above compound is readilyaccomplished by procedures known per se, such as, for example, bytreatment with anhydrous acid (HF) preferably in the presence ofanisole.

The strategy employed in the chemical synthesis of the protectedcarboxyl terminal tetradecapeptide I was as follows:

For the synthesis of the carboxyl terminal tripeptideBoc-Ala-Glu(OBzl)-Asn-OBzl, H-Glu(OBzl)-OH was first coupled toBoc-Ala-OSu to give the protected dipeptide fragmentBoc-Ala-Glu(OBzl)-OH which was then condensed with HCl . H-Asn-OBzl viathe DCC/HOSu procedure of Wunsch and Drees, Chem. Ber. 99, 110 (1966).The hydrochloride salt of asparagine benzyl ester was prepared fromBoc-Asn-OBzl which in turn was synthesized from commercially availableBoc-Asn-OH and benzyl bromide using the cesium salt of the amino acid.The Boc-protecting group was removed by a 30 minute treatment with 4 NHCl in dry THF.

Reaction between H-Glu(OBzl)-OH and Boc-Glu(OBzl)-OSu producedBoc-Glu(OBzl)-Glu(OBzl)-OH as a colorless clear oil. It was subsequentlyutilized in the synthesis of the pentapeptideBoc-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl in a DCC/HOSu mediatedfragment condensation using HCl . H-Ala-Glu(OBzl)-Asn-OBzl that wasderived from Boc-Ala-Glu(OBzl)-Asn-OBzl upon 4 N HCl/THF treatment. Theaforesaid protected pentapeptide was obtained in good yield as acrystalline pure material.

For the preparation of the octapeptideBoc-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl, therequired tripeptide Boc-Glu(OBzl)-Val-Val-OH was first prepared.Boc-Val-OSu was allowed to react with free valine to provideBoc-Val-Val-OH which on deblocking with 4 N HCl in THF followed byreaction with Boc-Glu(OBzl)-OSu yielded the desired tripeptide which wascrystallized as cyclohexylamine salt Boc-Glu(OBzl)-Val-Val-OH . CHA. Thecyclohexylamine salt was converted to the free acid and was then coupledby DCC in the presence of HOSu to HCl .H-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl that was derived fromBoc-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl on treatment with HCl inTHF. The protected octapeptideBoc-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl wasobtained in purified form as an amorphous solid.

For the synthesis of the undecapeptideBoc-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl,the required tripeptide fragment was synthesized starting fromBoc-Lys(Z)-OSu and H-Lys(Z)-OH. The dipeptide Boc-Lys(Z)-Lys(Z)-OH thusobtained was treated with 4 N HCl in THF and the ensuing salt HCl .H-Lys(Z)-Lys(Z)-OH was then allowed to react with Boc-Glu(OBzl)-OSu toprovide the desired tripeptide Boc-Glu(OBzl)-Lys(Z)-Lys(Z)-OH. Thetripeptide was then activated with DCC and HOSu according to theprocedure of Weygard et al, Z. Naturforsch, 21b, 426 (1966) and thesolution of the tripeptide active ester Boc-Glu(OBzl)-Lys(Z)-Lys(Z)-OSugenerated in situ was combined with the trifluoroacetate salt ofH-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl-Ala-Glu(OBzl)-Asn-OBzl derivedfrom the corresponding blocked octapeptide by a 30 minute treatment withTFA. The desired protected undecapeptideBoc-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu-(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzlwas thus obtained. Amino acid analysis and microanalysis gave theexpected values.

The synthesis of the protected tetradecapeptide followed a similarpattern. Boc-Leu-OSu was coupled to H-Lys(Z)-OH to provideBoc-Leu-Lys(Z)-OH. After removal of the N.sup.α -Boc-group with 4 N HClin THF and reaction with Boc-Asp(OBzl)-OSu the protected tripeptideBoc-Asp(OBzl)-Leu-Lys(Z)-OH was obtained as a crystalline pure solid. Itwas converted into the active ester Boc-Asp(OBzl)-Leu-Lys(Z)-OSu andcondensed with the trifluoroacetate salt ofH-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzlobtained from TFA treatment of the corresponding blocked undecapeptide.The desired productBoc-Asp(OBzl)-Leu-Lys(Z)-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzlwas obtained in good yield. Thin layer chromatography indicated that theproduct was homogeneous.

The protected amino terminal tetradecapeptideAc-Ser(Bzl)-Asp(OBzl)-Ala-Ala-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OHwas assembled using procedures well known in the peptide synthesis artfrom an acetyl tetrapeptide fragment, a hexapeptide, and anothertetradecapeptide. For the synthesis of the amino terminal acetyltetrapeptide, Ac-Ser(Bzl)-OBzl was prepared from Ac-OSu and thehydrochloride salt of H-Ser(Bzl)-OBzl. The same compound can also beprepared from Ac-Ser(Bzl)-OH and benzyl bromide via the cesium salt ofthe amino acid. Hydrazinolysis of Ac-Ser(Bzl)-OBzl gaveAc-Ser(Bzl)-HNNH₂ as a pure crystalline solid in good yield. Deblockingof Boc-Ala-Ala-OH afforded the dipeptide hydrochloride salt HCl .H-Ala-Ala-OH. Coupling of this dipeptide with Boc-Asp(OBzl)-OSu providedthe tripeptide Boc-Asp(OBzl)-Ala-Ala-OH which was isolated as thedicyclohexyl amine salt. Removal of the amino protecting group andcondensation with Ac-Ser(Bzl)-HNNH₂ via the azide procedure of Honzl andRudinger, Collection Czech. Chem. Commun. 26, 2333 (1961) gave thepartially protected tetrapeptide Ac-Ser(Bzl)-Asp(OBzl)-Ala-Ala-OH whichon reaction with equivalent amounts of hydrazine mediated by thedicyclohexylcarbodiimide in the presence of 1-hydroxybenzotriazoleproduced the desired intermediate Ac-Ser(Bzl)-Asp(OBzl)-Ala-Ala-HNNH₂.

For the synthesis of the hexapeptideBoc-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-HNNH₂, a processinvolving the stepwise elongation of the peptide chain from theC-terminal end was adopted. Thus, H-Glu(OBzl)-OH was coupled withBoc-Ser(Bzl)-OSu to form the protected dipeptideBoc-Ser(Bzl)-Glu(OBzl)-OH which on removal of the Boc-group and furtherreaction with Boc-Ser(Bzl)-OSu gave rise to the tripeptideBoc-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH. Deprotection of the amino groupfollowed by condensation of the resultant tripeptide saltHCl.H-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH with Boc-Thr(Bzl)-OSu yielded theprotected tetrapeptide Boc-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH whichon treatment with HCl in THF removed the Boc-group and further reactionwith Boc-Asp(OBzl)-OSu resulted in the formation of the protectedpentapeptide Boc-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH. TheBoc-group on this compound was similarly recovered with HCl treatmentand the ensuing product HCl .H-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH was subsequentlycoupled to Boc-Val-OSu to giveBoc-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH as acrystalline pure material.

The α-carboxyl group on the terminal glutamic acid residue of theaforesaid hexapeptide was then specifically converted into the hydrazidefunction by the reaction with an equivalent amount of hydrazine usingdicyclohexylcarbodiimide as coupling agent in the presence of1-hydroxybenzotriazole. The desired hexapeptide hydrazideBoc-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-HNNH₂ wasisolated as pure crystalline solid in a reasonable yield.

For the synthesis of the protected tetrapeptideBoc-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH, a similar stepwise procedure usingthe N-hydroxysuccinimide active ester procedure of Anderson et al, J.Amer. Chem. Soc. 86, 1839 (1964) was utilized. Reaction betweenBoc-Thr(Bzl)-OSu and H-Lys(Z)-OH gave the dipeptideBoc-Thr(Bzl)-Lys(Z)-OH as an oil which was deprotected at the α-aminoend and allowed to react with Boc-Thr(Bzl)-OSu to provide the tripeptideBoc-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH as a crystalline solid. Removal of theBoc-group and reaction of the resultant materialHCl.H-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH with Boc-Ile-OSu yielded the desiredprotected tetrapeptide Boc-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH as acrystalline pure compound after chromatography on a silica gel column.This tetrapeptide fragment was then deprotected at the amino terminaland condensed with the hexapeptideBoc-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-HNNH₂ by theazide method to produce the protected decapeptideBoc-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OHin good yield.

Removal of the Boc-group from the decapeptide compound with TFA andsubsequent coupling with the N-terminal tetrapeptideAc-Ser(Bzl)-Asp(OBzl)-Ala-Ala-HNNH₂ through the azide procedure resultedin formation of the required protected tetradecapeptideAc-Ser(Bzl)-Asp(OBzl)-Ala-Ala-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH.For the final coupling, This acetyl tetradecapeptide was activated withDCC and HOBT and the ensuing active ester was then allowed to react withTFA .H-Asp(OBzl)-Leu-Lys(Z)-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzlwhich was derived from the corresponding blocked compound on treatmentwith TFA to give the protected acetyl octacosapeptideAc-Ser(Bzl)-Asp(OBzl)-Ala-Ala-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-Asp(OBzl)-Leu-Lys(Z)-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl.Treatment with anhydrous HF removed all the protecting groups andpurification on ion-exchange chromatography yielded thymosin α₁.

[The synthesis of [Asn² ]-thymosinα₁ followed the same pattern as thesynthesis of thymosinα₁. All of the intermediates used were the sameexcept the N-terminal acetyl tetrapeptide hydrazideAc-Ser(Bzl)-Asn-Ala-Ala-HNNH₂. For the synthesis of this compound,Boc-Ala-Ala-OH was first converted into Boc-Ala-Ala-OBzl which onselective removal of the Boc-group with HCl gave the dipeptide estersalt HCl.H-Ala-Ala-OBzl. The dipeptide was then coupled with Boc-Asn-OHusing the DCC-HOBT procedure of Konig and Geiger, Chem. Ber. 103, 788(1970) to give the protected tripeptide ester Boc-Asn-Ala-Ala-OBzl whichwas treated with HCl in THF to remove the Boc-group. The resultantproduct HCl.H-Asn-Ala-Ala-OBzl was then condensed (DCC-HOBT procedure)with Ac-Ser(Bzl)-OH.DCHA to afford the desired tetrapeptideAc-Ser(Bzl)-Asn-Ala-Ala-OBzl.

The corresponding hydrazide Ac-Ser(Bzl)-Asn-Ala-Ala-HNNH₂ was obtainedin good yield on hydrazinolysis of this compound. Fragment condensationbetween the hydrazide and the decapeptideTFA.H-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OHproduced the protected tetradecapeptideAc-Ser(Bzl)-Asn-Ala-Ala-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH.This compound was then coupled to the deblocked C-terminaltetradecapeptide TFA salt discussed above to give the correspondingprotected [Asn² ]-thymosinα₁. Removal of all the protecting groups bytreatment with anhydrous HF followed by column chromatographypurification gave the desired analog [Asn² ]-thymosinα₁.]

In a further aspect of this invention it has been found that several ofthe intermediate peptides used in the above synthesis of thymosinα₁[and[Asn² ]-thymosinα₁ ] have activity in the regulation,differentiation and function of T-cells.

Thus, the protected octapeptideBoc-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl can bedeprotected by hydrogenolysis followed by treatment with trifluoroaceticacid in the usual manner to give the free octapeptideGlu-Val-Val-Glu-Glu-Ala-Glu-Asn. This product is purified byion-exchange column chromatography to yield material homogeneous on thinlayer chromatography and paper electrophoresis.

Similarly, deprotection of the protected undecapeptideBoc-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzlwith anhydrous hydrofluoric acid provided the free undecapeptideGlu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn which was homogeneous onpaper electrophorisis after ion-exchange column chromatography.

The carboxy terminal tetradecapeptideAsp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn was obtained inlike manner from the protected tetradecapeptideBoc-Asp(OBzl)-Leu-Lyz(Z)-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzlby anhydrous hydrofluoric acid treatment and purification on an ionexchange column.

The amino terminal tetradecapeptideAc-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-OH wasobtained in like manner from the protected tetradecapeptide.Ac-Ser(Bzl)-Asp(OBzl)-Ala-Ala-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OHby anhydrous hydrofluoric acid treatment and purification on an ionexchange column.

Thymosinα₁, [[Asn² ]-thymosinα₁,] or the aforesaid novel octa-, undeca-or tetradecapeptides which form a part of the present invention, may beadministered to warm blooded mammals by parenteral application eitherintravenously, subcutaneously or intramuscularly. These compounds arepotent immunopotentiating agents with a daily dosage in the range ofabout 1 to 100 mg/kg of body weight per day for intravenousadministration. Obviously the required dosage will vary with theparticular condition being treated, the severity of the condition andthe duration of treatment. A suitable dosage form for pharmaceutical useis 1 mg. of lyophilized thymosinα₁, [[Asn² ]-thymosinα₁,] or one of theaforesaid peptide fragments thereof to be reconstituted prior to use bythe addition of sterile water or saline.

Also included within the scope of the present invention are thepharmaceutically acceptable salts of thymosinα₁, [[Asn² ]-thymosinα₁,]and the aforesaid peptide fragments thereof. Suitable salts includesodium, potassium or a strong organic base such as guanidine. Inaddition, the counter ions of these cations such as the chloride,bromide, sulfate, phosphate, maleate, acetate, citrate, benzoate,succinate, malate, ascorbate and the like, may be included in thepreparation.

Abbreviations used herein have the following meaning: Boc,t-butyloxycarboxy; Bzl, benzyl; DCC, dicyclohexylcarbodiimide; DMF,dimethylformamide; THF, tetrahydrofuran; HOSu, N-hydroxysuccinimide;Triton B, 40% methanolic solution of trimethylbenzylammonium hydroxide;NMM, N-methylmorpholin; CHA, cyclohexylamine; DCHA, dicyclohexylamine;Z, benzyloxycarbonyl; Cl₂ 7,2,4-dichlorobenzyloxycarbonyl; DMSO,dimethylsulfoxide; and TFA, trifluoroacetic acid; TLC, thin layerchromatography.

While specific protecting groups have been employed in describing thepreferred embodiment synthesis of thymosinα₁ [and [Asn² ]-thymosinα₁ ],it is within the skill of the art to utilize equivalent conventionalprotecting groups in such synthesis.

In a broader aspect of the present invention, --Ser(R¹) is utilized asthe protected form of serine wherein R¹ is a conventional protectinggroup for the hydroxyl group of the serine residues selected frombenzyl, acetyl, benzoyl, tert-butyl, trityl, 4-bromobenzyl,2,6-dichlorobenzyl and benzyloxycarbonyl; --Asp(OR²) is utilized as theprotected form of aspartic acid wherein R² is a conventional protectinggroup for carboxyl groups selected from esters such as aryl esters,particularly phenyl or phenyl substituted with lower alkyl, halo, nitro,thio or substituted thio, i.e., methylthio, aralkyl esters such asbenzyl or benzyl substituted with methoxy, halo or nitro, lower alkylesters such as methyl, ethyl, tert-butyl and tert-amyl, substitutedlower alkyl esters such as 2-haloethyl, β,β-dimethylaminoethyl andcyanomethyl, benzhydryl esters and phenacyl esters; --Thr(R³)-- isutilized as the protected form of threonine wherein R³ is a conventionalprotecting group for the hydroxyl group of the threonine residues suchas benzyl, acetyl, benzoyl, tert-butyl, trityl, 2,6-dichlorobenzyl,4-bromobenzyl and benzyloxycarbonyl; --Glu(OR⁴)-- is utilized as theprotected form of glutamic acid wherein R⁴ is independently selectedfrom the protecting groups set forth for R² above; --Lys(R⁵)-- isutilized as the protected form of lysine wherein R⁵ is a conventionalω-amino protecting group selected from benzyloxycarbonyl which may beoptionally substituted in the aromatic ring such as by 4-chloro,2-bromo, 4-bromo, 2,4-dichloro, 4-nitro, 4-methoxy, 3,5-dimethoxy,4-methyl, 2,4,6-trimethyl, 4-phenylazo, 4-(4-methoxyphenylazo),2-(N,N-dimethylcarbonamido), 4-dihydroxyboryl, and2-nitro-4,5-dimethoxy, urethane type protecting groups such as4-toluenesulfonylethyloxycarbonyl, 9-fluorenylmethyloxycarbonyl andrelated base cleavable groups, 5-benzisoxazolylmethyleneoxycarbonyl,methylthio- and methylsulfonylethyloxycarbonyl, isonicotinyloxycarbonyl,haloethyloxycarbonyl, diisopropylmethyloxycarbonyl,benzhydryloxycarbonyl, isobornyloxycarbonyl,dinitrodiphenylmethyloxycarbonyl, tert.butyloxycarbonyl,tert.--amyloxycarbonyl, adamantyloxycarbonyl, cyclopentyloxycarbonyl,methylcyclobutyloxycarbonyl, methylcyclohexyloxycarbonyl,2-arylisopropyloxycarbonyl groups such as2-(p-biphenylyl)-isopropyloxycarbonyl, 2-(4-pyridyl)isopropyloxycarbonyland related nitrogen containing urethane groups; acyl groups such asformyl, trifluoroacetyl, phthaloyl, benzenesulfonyl, acetoacetyl,chloroacetyl, 2-nitrobenzoyl, 4-toluenesulfonyl, sulfenyl groups such asbenzenesulfenyl, o-nitrophenylsulfenyl and related sulfenyl groups, andaryl-lower alkyl groups such as diphenylmethyl and triphenylmethyl; and--Asn-OR⁶ is utilized as the protected form of asparagine wherein R⁶ isa conventional carboxyl protecting group which is independently selectedfrom the protecting groups set forth for R² above.

In a further embodiment of the present invention, thymosinα₁, wasprepared by solid phase peptide synthesis. In such embodimentcommercially available benzhydrylamine resin was neutralized andacylated with N.sub.α -Boc-α-benzyl-L-aspartic acid in the presence ofdicyclohexylcarbodiimide to give Boc-asparaginyl resin. This amino acidresin was then benzoylated according to the procedure of Wang, J. Amer.Chem. Soc., 95, 1328 (1973) and placed into the reaction vessel of anautomated peptide synthesis apparatus. The machine was then programmedto perform the solid phase synthesis to incorporate the following aminoacid unit in each cycle sequentially: Boc-Glu(OBzl)-OH, Boc-Ala-OH,Boc-Glu(OBzl)-OH, Boc-Glu(OBzl)-OH, Boc-Val-OH, Boc-Val-OH,Boc-Glu(OBzl)-OH, Boc-Lys (Cl₂ Z)-OH, Boc-Lys(Cl₂ Z)-OH,Boc-Glu(OBzl)-OH, Boc-Lys (Cl₂ Z-OH, Boc-Leu-OH, Boc-Asp(OBzl)-OH,Boc-Lys (Cl₂ Z)-OH, Boc-Thr(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Ile-OH,Boc-Glu(OBzl)-OH, Boc-Ser(Bzl)-OH, Boc-Ser(Bzl)-OH, Ber-Thr(Bzl)-OH,Boc-Asp(OBzl)-OH, Boc-Val-OH,2x Boc-Ala-OH, Boc-Asp(OBzl)-OH,Boc-Ser(Bzl)-OH and CH₃ COOH. Four-fold excess of each Boc-Amino acidand dicyclohexylcarbodiimide were used in each coupling reaction (120min) and a 30 min treatment with 33% trifluoroacetic acid in CH₂ Cl₂ wasused as deprotecting agent for the Boc-group. The acetyl protectedoctacosapeptide resin of the formulaAc-Ser(Bzl)-Asp(OBzl)-Ala-Ala-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl-Glu(OBzl)-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Cl₂Z)-Asp(OBzl)-Leu-Lys(Cl₂ Z)-Glu(OBzl)-Lys(Cl₂ Z)-Lys(Cl₂Z)-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asp(OBzl)CO-NH-CH(C₆ H₅) ##STR1## Resin thus obtained was then treated withanhydrous HF-anisole to remove all of the side chain protecting groupsand at the same time to release the unprotected peptide from the resin.The crude material was desalted on a Sephadex G-10 column and thenpurified on a DEAE-Sephadex A-25 column eluted with a linear gradient ofsodium chloride in pH7.0 Tris HCl buffer. The purified thymosinα₁product gave satisfactory amino acid analyses and behaved identically tothe natural thymosinα₁ on thin layer electrophoresis. It contained avery small contaminant which migrated faster than synthetic thymosinα₁prepared by the solution phase method. While specific protecting groupshave been disclosed in regard to the solid phase synthesis, it should benoted that each amino acid can be protected by any of the protectivegroups discribed for the respective amino acids in solution phasesynthesis.

The present invention is further illustrated by the Examples whichfollow.

EXAMPLE 1 Boc-Asn-OBzl

Boc-Asn-OH (11.0 g, 47.5 mmol) was dissolved in 200 ml of MeOH and 20 mlof water was added. The solution was titrated to pH 7.0 (pH paper) witha 20% aq. solution of Cs₂ CO₃ (˜55 ml). The mixture was evaporated todryness and the residue reevaporated twice from DMF (120 ml each, 45°).The white solid obtained was then stirred with 8.9 g of benzyl bromide(52 mmol) in 120 ml DMF for 6 hours. On evaporation to dryness andtreatment with a large volume of water, the product solidifiedimmediately. It was collected by filtration, dissolved in ethyl acetate,washed with water, dried over Na₂ SO₄, evaporated to a solid mass andcrystallized from ethyl acetate with petroleum ether:

Yield, 13.8 g (90.3%); mp 120°-122°; [α]_(D) ²⁵ =-17.29° (c 1, DMF).

Anal. Calcd for C₁₆ H₂₂ N₂ O₅ (322.36): C, 59.61; H, 6.88; N, 8.69.Found: C, 59.76; H, 6.81; N, 8.82.

EXAMPLE 2 Boc-Ala-Glu(OBzl)-OH

H-Glu(OBzl)-OH (7.0 g, 29.5 mmol) was finely ground in a mortar andpestle and then stirred with 8.88 g (32.3 mmol) of Boc-Ala-OSu for 48hours in 250 ml DMF in the presence of 6 ml N-methylmorpholine (NMM).Some more NMM was added to maintain the reaction slightly basic duringthe reaction. The solvent was evaporated and the residue partitionedbetween 300 ml ethyl acetate and 500 ml H₂ O containing 2 ml of 10% H₂SO₄. The organic layer was then washed three times with water, dried(Na₂ SO₄) and evaporated to dryness. The product was taken up in a smallvolume of ether and treated with a large volume of petroleum ether. Awhite amorphous solid was obtained which was homogeneous on tlc:

Yield, 11.0 g (91.5%); mp 84°-88°; [α]_(D) ²⁵ =-8.08° (c 1, DMF).

Anal. Calcd for C₂₀ H₂₈ N₂ O₇ (408.44): C, 58.81; H, 6.91; N, 6.85.Found: C, 58.89; H, 6.91; N, 6.89.

EXAMPLE 3 Boc-Ala-Glu(OBzl)-Asn-OBzl

Boc-Asn-OBzl (13.7 g, 42.4 mmol) was dissolved in 80 ml of THF andtreated with 500 ml of 4 N HCl in THF. The mixture was left standing for45 minutes during which time some product began to precipitate. Ontreatment with 1000 ml of ether, a white solid material formedimmediately. The product was filtered and washed with ether and driedover NaOH pellets in vacuo:

Yield, 10.3 g (94%); mp 122°-126°; [α]_(D) ²⁵ =+6.82°; tlc homogeneous.

Anal. Calcd for C₁₁ H₁₅ N₂ O₃ Cl 258.71): C, 51.07; H, 5.84; N, 10.83.Found: C, 50.45; H, 5.96; N, 10.69.

Boc-Ala-Glu(OBzl)-OH (10.4 g, 25.4 mmol), HCl H-Asn-OBzl (6.56 g, 25.4mmol) and HOSu (5.9 g, 50.8 mmol) were dissolved in DMF (250 ml, 0°).DCC (5.7 g, 27.6 mmol) was added followed immediately by Et₃ N (3.5 ml).The mixture was stirred at 0° for 2 hours and then at 25° for 40 hoursduring which period some more Et₃ N was added from time to time tomaintain the reaction slightly basic. The insoluble by-products formedwere filtered off and the filtrate evaporated to dryness. The residualoily material solidified on treatment with water. The crude product wastaken up in CHCl₃, washed with water (3×), dried over Na₂ SO₄ andevaporated to a smaller volume. Some solid formed at this stage wasfiltered off (heavily contaminated with dicyclohexylurea) and thefiltrate treated with petroleum ether. A crystalline product wasobtained: Yield, 8.0 g (51.4%); mp 102°-105°; [α]_(D) ²⁵ =-12.05° (c 1,DMF).

Anal. Calcd for C₃₁ H₄₀ N₄ O₉ (612.66); C, 60.76; H, 6.58; N, 9.14.Found: C, 60.91; H, 6.68; N, 9.24.

EXAMPLE 4 Boc-Glu(OBzl)-Glu(OBzl)-OH

H-Glu(OBzl)-OH (4.74 g, 20 mmol) was ground in a mortar and pestle andstirred with Boc-Glu(OBzl)-OSu (8.7 g, 20 mmol) in DMF for 36 hours inthe presence of 3.6 ml NMM. The ensuing solution was evaporated to asyrup and treated with water. The oily precipitate was taken up in ethylacetate, washed successively with 5% HOAc and water (3x), dried over Na₂SO₄ and evaporated to dryness yielding 14.03 g of a clear oil. It wasleft standing submerged under petroleum ether. The residual oil weighed10.2 g (90.0%). TLC indicated that the product was homogeneous. [α]_(D)²⁵ =-7.59° (c 1, DMF).

Anal. Calcd for C₂₉ H₃₆ N₂ O₉ (556.60): C, 62.57; H, 6.52; N, 5.03.Found: C, 62.37; H, 6.34; N, 5.01.

EXAMPLE 5 Boc-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl

Boc-Ala-Glu(OBzl)-Asn-OBzl (28.2 g; 46 mmol) was treated with 1.1 literof 4 N HCl in THF for 1 hour. Evaporation of the solvent and excess acidleft an oil which was evaporated twice more with fresh THF. The residualoil turned into a solid when treated with a large volume of ether. Thesolid HCl.H-Ala-Glu(OBzl)-Asn-OBzl was stirred withBoc-Glu(OBzl)-Glu(OBzl)-OH (25.6 g, 46 mmol), HOSu (10.6 g, 92 mmol) andDCC (10.9 g, 53 mmol) in DMF (540 ml) at 0° for 1 hour and then at 25°for 48 hours. Et₃ N was added to maintain the reaction slightly basicover the entire period of time (˜16 ml Et₃ N total). The insolubleby-products formed were filtered off and the filtrate evaporated todryness. The crude product was dissolved in CHCl₃, washed with water(3x), dried over Na₂ SO₄ and evaporated to dryness. The productsolidified when treated with petroleum ether. Recrystallized fromi-PrOH:

Yield, 28.9 g (59.8%); mp 169°-175°; [α]_(D) ²⁵ =-11.78° (c 1, DMF).

Anal. Calcd for C₅₅ H₆₆ N₆ O₁₅ (1051.13): C, 62.85; H, 6.33; N, 8.00.Found: C, 62.57; H, 6.35; N, 8.06.

EXAMPLE 6 Boc-Val-Val-OH

Boc-Val-OSu (12.6 g, 40 mmol) and H-Val-OH (4.68 g, 40 mmol) werecondensed in DMF (250 ml) for 96 hours in the presence of 2 ml Et₃ N.More Et₃ N was added when needed to maintain the reaction slightlybasic. The remaining insoluble material was filtered off and thefiltrate evaporated to dryness (45°). The residue was partitionedbetween ether and dilute H₂ SO₄ (˜1%) and the organic layer washed withwater (3x), dried over Na₂ SO₄ and evaporated to a foamy glass. Theproduct was crystallized from ether and petroleum ether:

Yield, 12.2 g (96.4%); mp 155°-158°; [α]_(D) ²⁵ =+1.10° (c 1, DMF).

Anal. Calcd for C₁₅ H₂₈ N₂ O₅ (316.4): C, 56.94; H, 8.92; N, 8.85.Found: C, 56.64; H, 9.16; N, 8.71.

EXAMPLE 7 Boc-Glu(OBzl)-Val-Val-OH.CHA

Boc-Val-Val-OH (40.5 g, 128 mmol) was treated with 1.8 l of 4 N HCl inTHF for 60 minutes. Evaporation to remove excess acid and solventfollowed by treatment with ether provided 34.5 g of HCl.H-Val-Val-OH asa white amorphous powder. It was treated with Boc-Glu(OBzl)-OSu (55.6 g,128 mmol) in 1 liter DMF for 24 hours in the presence of 54 ml Et₃ N.The reaction mixture was filtered to remove some insoluble material andthe filtrate evaporated to dryness. The remaining oily residue was takenup in EtOAc (1.5 l) and washed with 5% HOAc (2x) followed by water (3x).The organic layer was dried (Na₂ SO₄) and evaporated to dryness to givea colorless clear oil which did not crystallize. It was thus dissolvedin 3.2 l of ether and treated with cyclohexylamine (CHA) (17 ml) untilthe pH (moist pH paper) of the mixture was 7.5. The solid salt obtainedwas collected and recrystallized from MeOH and ether:

Yield, 58.9 g (72.7%); mp 158°-60°; [α]_(D) ²⁵ =-33.41° (c 1, MeOH).

Anal. Calcd for C₃₃ H₅₄ N₄ O₈ (634.79): C, 62.44; H, 8.57; N, 8.83.Found: C, 62.18; H, 8.37; N, 8.77.

EXAMPLE 8 HCl.H-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl

Boc-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl (3.9 g, 3.48 mmol) wastreated with 15 ml 4 N HCl in THF for 30 minutes. Some crystallineproduct started to form. Ether (210 ml) was added and the precipitatedsolid was collected and washed with ether. The crude material wascrystallized from MeOH and ether:

Yield, 2.58 g (75.1%); mp 148°-151°; [α]_(D) ²⁵ =-3.65° (c 1, DMF).

Anal. Calcd for C₅₀ H₅₈ N₆ O₁₃.HCl (987.48): C, 60.82; H, 6.02; N, 8.51.Found: C, 60.02; H, 6.18; N, 8.37.

EXAMPLE 9Boc-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu-(OBzl)-Asn-OBzl

Boc-Glu(OBzl)-Val-Val-OH.CHA (1.69 g, 2.66 mmol) was suspended in water(40 ml) and ethyl acetate (40 ml) in a separatory funnel when 4 ml of 1M H₂ SO₄ was added. After vigorous shaking, the solid dissolved and theorganic layer was washed several times with water, dried over Na₂ SO₄and evaporated to an oil (1.45 g). The free tripeptide thus obtained wasthen condensed with 2.58 g ofHCl.H-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl (2.61 mmol) in 15 mlDMF in the presence of HOSu (0.612 g, 5.32 mmol), NMM (0.3 ml, 2.66mmol) and DCC (0.63, 3.06 mmol) during 1 hour at 0° and 60 hours at 25°.More NMM was added when needed to maintain the reaction slightly basic.An insoluble by-product was formed which was filtered off and thefiltrate evaporated to dryness (45°). The remaining oily residuesolidified when treated with water. The crude solid was dissolved in DMF(50 ml) and precipitated with MeOH (300 ml):

Yield, 2.25 g (58.7%); mp 277°-280°; [α]_(D) ²⁵ =-12.43° (c 1, DMF).

Anal. Calcd for C₇₇ H₉₇ N₉ O₂₀ (1468.62): C, 62.97; H, 6.66; N, 8.58.Found: C, 62.72; H, 6.71; N, 8.53.

EXAMPLE 10 Boc-Lys(Z)-Lys(Z)-OH.DCHA

Boc-Lys(Z)-OH (15 g, 39.5 mmol) was stirred with HOSu (5.8 g, 50.5 mmol)and DCC (8.66 g, 42 mmol) in THF (250 ml) for 3 hours. An insolubleby-product was filtered off and the filtrate evaporated to dryness. Theresidual syrup (24.2 g) was treated with i-PrOH (150 ml) and petroleumether (150 ml) to yield an oily product (21 g) which failed tocrystallize. The crude active ester Boc-Lys(Z)-OSu was thus used forcondensation with H-Lys(Z)-OH (10.6 g, 38 mmol) in DMF (250 ml) for 72hours in the presence of 5.5 ml Et₃ N. More Et₃ N was added occasionallyin order to maintain the stirred reaction mixture slightly basic. Somesmall quantity of undissolved material was then filtered off and thefiltrate evaporated to dryness (45°). The remaining oily residue wastreated with 1 liter of 5% HOAc. The product precipitated was extractedinto ethyl acetate and the organic phase washed with water, dried overNa₂ SO₄ and evaporated to an oil. It was crystallized from ethyl acetate(300 ml) containing DCHA (10 ml) as a salt. Recrystallized from MeOH andether:

Yield, 22.7 g (72.5%); mp 160°-162°; [α]_(D) ²⁵ =-2.21° (c 1, MeOH).

Anal. Calcd for C₄₅ H₆₉ N₅ O₉ (824.04): C, 65.59; H, 8.44; N, 8.50.Found: C, 65.40; H, 8.50; N, 8.39.

EXAMPLE 11 Boc-Glu(OBzl)-Lys(Z)-Lyz(Z)-OH

Boc-Lys(Z)-Lys(Z)-OH.DCHA (10 g, 12.14 mmol) was partitioned betweenEtOAc (1 liter) and 0.1 N H₂ SO₄ (1 liter). The organic layer was thenwashed with water (3x), dried over Na₂ SO₄ and evaporated to dryness(7.9 g). The free acid, Boc-Lys(Z)-Lys(Z)-OH, thus obtained was treatedwith freshly prepared 4 N HCl in THF for 30 minutes. The solvent and theexcess acid was evaporated (30°) and the residue re-evaporated twicewith THF. The remaining residue solidified when treated with ether. Thesalt HCl.H-Lys(Z)-Lys(Z)-OH was collected by filtration and washedseveral times with ether to yield 6.7 g of white powder. It wasdissolved in DMF (70 ml), chilled in an ice-bath and treated with Et₃ N(1.63 ml) followed by Boc-Glu(OBzl)-OSu (5.54 g, 12.76 mmol). Themixture was stirred at 0° for 1 hour and then at 25° for 24 hours. MoreEt₃ N was added during this time to maintain the reaction atapproximately pH 7.5 (pH paper). A few ml of acetic acid was added tomake the reaction acidic (pH 3.5) and the solvent removed byevaporation. The ensuing residue was taken up in EtOAc, washed withwater (3x), dried over Na₂ SO₄ and evaporated to dryness when theproduct began to solidify. It was triturated in ether and recrystallizedfrom ethyl acetate:

Yield, 7.26 g (69.5%); mp 153°-155°; [α]_(D) ²⁵ =-2.71° (c 1, THF).

Anal. Calcd for C₄₅ H₅₉ N₅ O₁₂ (861.96): C, 62.70; H, 6.90; N, 8.12.Found: C, 62.51; H, 6.93; N, 8.13.

EXAMPLE 12Boc-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl

Boc-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl. (1.7g, 1.16 mmol) was treated with TFA (24 ml) for 30 minutes. Afterevaporation of the excess acid (30°) the residue was triturated withether. The powder obtained was washed thoroughly with ether andpetroleum ether and dried over NaOH in vacuo to give thetrifluoroacetate salt of the octapeptide (1.71 g). The active esterBoc-Glu(OBzl)-Lys(Z)-Lys(Z)-OSu was then generated in situ by stirringBoc-Glu(OBzl)-Lys(Z)-Lys(Z)-OH (0.998 g, 1.16 mmol), HOSu (0.16 g, 1.4mmol) and DCC (0.274 g, 1.33 mmol) in 15 ml DMF at 0° for 3 hours. Tothis solution containing the tripeptide active ester, the octapeptidesalt CF₃COOH.H-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl(1.71 g) was added together with 0.2 ml of Et₃ N. A few more drops ofEt₃ N and DMF (15 ml) were added and the reaction was stirred for 3 daysat 25°. A gelatinous semi-solid formed. It was acidified with aceticacid and treated with water. The white solid precipitate was collectedand washed (H₂ O, MeOH, Ether) to yield 2.25 g of crude product meltingat 310°-313°. It was dissolved in DMF and precipitated with MeOH:

Yield, 1.75 g (68.3%); mp 314°-316°; [α]_(D) ²⁵ =-13.78° (c 1, DMSO);homogeneous on tlc.

Anal. Calcd for C₁₁₇ H₁₄₆ N₁₄ O₂₉ (2212.45): C, 63.51; H, 6.65; N, 8.86.Found: C, 63.31; H, 6.78; N, 8.87.

Amino Acid Anal. Asp, 1.06; Glu, 5,00; Ala, 1.06; Val, 2.15; Lys, 1.87;NH₃, 1.06.

EXAMPLE 13 Boc-Leu-Lys(Z)-OH.DCHA

Boc-Leu-OSu (4.0 g, 12.2 mmol) and H-Lys(Z)-OH (3.42 g, 12.2 mmol) werecondensed in DMF (75 ml) during 48 hours in the presence of Et₃ N (1.7ml). The reaction pH was maintained at 7.5 (moist pH paper) by additionof Et₃ N periodically as usual. The remaining insoluble material wasfiltered off and the filtrate evaporated to dryness. The ensuing foamyglass was dissolved in ether (200 ml) and the mixture treated with 3 mlof DCHA to produce crystalline material which was collected, washed withether and recrystallized from MeOH and ether:

Yield, 5.7 g (69.5%); mp 140°-142°; [α]_(D) ²⁵ =-7.20° (c 1, MeOH).

Anal. Calcd for C₃₇ H₆₂ N₄ O₇ (674.90): C, 65.85; H, 9.26; N, 8.30.Found: C, 65.80; H, 9.07; N, 8.15.

EXAMPLE 14 Boc-Asp(OBzl)-Leu-Lys(Z)-OH

Boc-Leu-Lys(Z)-OH.DCHA (2.97 g, 4.4 mmol) was converted into the freeacid (partitioned between EtOAc and 0.1 N H₂ SO₄) and the colorless oilobtained (2.2 g) was treated with 4 N HCl in THF (40 ml) for 30 minutes.The excess acid and the solvent were evaporated (30°) and the residuetreated with ether. The remaining oil was dissolved in ether andevaporated twice more with fresh ether. The residue was then stirredwith Boc-Asp(OBzl)-OSu (1.85 g, 4.4 mmol) in the presence of Et₃ N (1.85ml) overnight. The reaction mixture was then evaporated to drynessgiving an oily residue which was taken up in ethyl acetate, washed withwater (3x), dried over Na₂ SO₄ and evaporated to dryness again. Thecrude product thus obtained was crystallized from ethyl acetate andpetroleum ether:

Yield, 1.52 g (49.6%); mp 109°-111°; [α]_(D) ²⁵ =-16.14° (c 1, DMF).

Anal. Calcd for C₃₆ H₅₀ N₄ O₁₀ (698.91): C, 61.88; H, 7.21; N, 8.02.Found: C, 61.72; H, 7.17; N, 8.01.

EXAMPLE 15Boc-Asp(OBzl)-Leu-Lys(Z)-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val-Val-Glu(OBzl)-Gl(OBzl)-Ala-Glu(OBzl)-Asn-OBzl

Boc-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl(1.2 g, 0.545 mmol) was treated with 35 ml TFA for 30 minutes. Theexcess acid was quickly evaporated and the residue triturated with etherseveral times to give 1.2 g of undecapeptide TFA-salt as a white powder.It was dissolved in a mixture of DMF (5 ml) and DMSO (2 ml) and treatedwith Boc-Asp(OBzl)-Leu-Lys(Z)-Osu generated in situ by stirringBoc-Asp(OBzl)-Leu-Lys(Z)-OH (0.381 g, 0.545 mmol) with HOSu (0.126 g,1.1 mmol) and DCC (0.124 g, 0.599 mmol) in 3 ml DMF at 0° for 3 hours.The mixture containing the tripeptide active ester and undecapeptide wasstirred at 0° for 2 hours and then at 25° for 3 days, during whichperiod Et₃ N was added from time to time in order to maintain the pHslightly basic. A gelatenous substance formed. It was triturated with 5%HOAc and the resulting white solid was filtered and washed with water,MeOH and ether to give 1.28 g of crude material melting at 325°-326°.Reprecipitation from DMF-DMSO (10 ml and 5 ml) and MeOH (230 ml) yielded1.22 g (80.2%); mp 326°-327°; [α]_(D) ²⁵ =-15.71° (C 1, DMF-DMSO).

Anal. Calcd for C₁₄₈ H₁₈₆ N₁₈ O₃₆ (2793.21): C, 63.64; H, 6.71; N, 9.03.Found: C, 62.78; H, 6.75; N, 9.04.

Amino Acid Anal. Asp, 2.00; Glu, 5.65; Ala, 1.12; Val, 2.24; Leu, 0.84;Lys, 3.00; NH₃, 1.02.

EXAMPLE 16 Glu-Val-Val-Glu-Glu-Ala-Glu-Asn

Boc-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl (0.72g, 0.49 mmol) was hydrogenated over 5% Pd/BaSO₄ (0.5 g) for 3 hours at50 psi in DMF-MeOH-H₂ O (40 ml-30 ml-2 ml). The mixture was thenfiltered and the filtrate evaporated to dryness. It was subsequentlytreated with 5 ml of TFA for 30 minutes and the residue obtained afterevaporation of the acid was triturated several times with ether. Theensuing white solid was taken up in water (20 ml) and lyophilized togive 0.47 g of crude product. The compound was loaded on a Bio-Rad AGl-X2 column (3×32 cm) equilibrated with pH 8.1 ammonium acetate buffer (2%HOAc made to pH 8.1 with NH₃). The column was eluted successively with200 ml each of 0.025 M pH 5.5 NH₄ OAc, 0.025 M HOAc, 0.05 M HOAc, 0.1 MHOAc, 0.25 M HOAc, 0.5 M HOAc, 0.75 M HOAc, 1 M HOAc. Fractions of 12 mlwere collected and the eluate from each tube was monitored by thin layerchromatogram. The fractions containing the desired material (tube225-229) were pooled and lypholized twice to yield 0.223 g (48.1%) ofthe pure product. Homogeneous on tlc and paper electrophoresis.

Anal. Calcd for C₃₇ H₅₉ N₉ O₁₈.3H₃ O (971.96): C, 45.71; H, 6.74; N,12.97. Found: C, 45.57; H, 6.83; N, 12.51.

Amino Acid Anal. Asp, 1.00; Glu, 3.95; Ala, 1.00; Val, 1.91; NH₃, 1.00.

EXAMPLE 17 Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn

Boc-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl (0.5 g, 0.226 mmol) was dissolved in 2 ml of TFA andstirred with 15 ml of HF at 0° for 15 minutes. After evaporation ofexcess acid (0°), the residue was dissolved in 5% aqueous HOAc, washedwith ether (3x), evaporated to a smaller volume and lyophilized to yield0.34 g of crude product. It was chromatographed on the ion-exchangecolumn as described above (see Example 16) to give 0.13 g (42.1%) ofpure material: [α]_(D) ²⁵ =-85.65° (c 1, H₂ O).

Anal. Calcd for C₅₄ H₉₀ N₁₄ O₂₃.4H₂ O (1375.43): C, 47.15; H, 7.18; N,14.25. Found: C, 47.03; H, 7.03; N, 13.88.

Amino Acid Anal. Asp, 1.03; Glu, 5.03; Ala, 0.94; Val, 2.06; Lys, 2.05;NH₃, 1.06.

EXAMPLE 18 Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn

Boc-Asp(OBzl)-Leu-Lys(Z)-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val-Val-Gl(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl(1.128 g, 0.404 mmol) was mixed with 7 ml of anisole and treated with 25ml anhydrous HF at 0° for 15 minutes. The excess acid was evaporated(0°) and the remaining residue partitioned between ether and water. Thewater layer was washed twice with ether, evaporated to 1/2 the originalvolume and lypholized to provide 0.69 g of crude material. It waschromatographed in the manner described above for compounds in Examples16 and 17. The material eluted at tubes 101-120 was collected andlyophilized to give 0.25 g of product which was shown to be slightlycontaminated with minor impurities. It was thus re-chromatographed onthe same column to give 0.155 g (22%) of pure product. Paperelectrophoresis indicated it was homogeneous. [α]_(D) ²⁵ =-86.27° (c 1,0.1 N HCl).

Anal. Calcd for C₇₀ H₁₁₈ N₁₈ O₂₈.5H₂ O (1749.86): C, 48.04; H, 7.37; N,14.41. Found: C, 47.90; H, 7.53; N, 14.49.

Amino Acid Anal. Asp, 1.98; Glu, 5.09; Ala, 1.04; Val, 2.02; Leu, 0.97;Lys, 2.89; NH₃, 1.09.

EXAMPLE 19 Ac-Ser(Bzl)-OH.DCHA

H-Ser(Bzl)-OH (18.6 g, 95.4 mmol) was dissolved in 45 ml of Triton B(40% methanolic solution of Trimethylbenzylammonium Hydroxide),evaporated to dryness and the residue re-evaporated twice with DMF (250ml each). The oily residue was taken up in DMF (250 ml) and stirred withAc-OSu (16.5 g, 95.4 mmol) for 5 hr. A few ml (˜5 ml) of NMM was addedto bring the reaction to slightly basic conditions during this period oftime. Acetic acid (˜5 ml) was then added and the solvents removed byevaporation, leaving an oily residue which was partitioned between 5%HOAc and EtOAc. The aqueous layer was extracted once more with EtOAc andthe combined EtOAc solution washed twice with small volumes of H₂ O,dried over Na₂ SO₄, and evaporated to approximately 300 ml (1/3 oforiginal volume). Crystalline product formed immediately when DCHA wasadded to a slightly basic condition (˜35 ml). The crude material (32.5g, mp 135°- 145°) was recrystallized from EtOH (150 ml) and ether (900ml):

Yield, 17.8 g (44.7%); mp 166°-168°; [α]_(D) ²⁵ =+34.18° (c 1, MeOH).

Anal. Calcd for C₂₄ H₃₈ N₂ O₄ (418.56): C, 68.87; H, 9.15; N, 6.69.Found: C, 68.54; H, 9.38; N, 6.85.

EXAMPLE 20 HCl.H-Ser(Bzl)-OBzl

Boc-Ser(Bzl)-OH (6.3 g, 21.4 mmol) was dissolved in MeOH (75 ml) and H₂O (7.5 ml). To this solution, 20% Cs₂ CO₃ was added until neutral (pH7.0; 28 ml) and then the solvents were removed by evaporation. Theresidue obtained was re-evaporated twice with DMF (50 ml each) and thesalt was stirred with benzyl bromide (4.28 g, 25 mmol) in 25 ml DMF for15 hr. Evaporation of the solvent gave an oily residue which wasextracted into EtOAc, washed with H₂ O (3 times), dried over Na₂ SO₄,and evaporated to dryness leaving a colorless clear oil (8.9 g). Thecompound did not crystallize at this stage. It was thus converted intothe HCl salt by deprotection of the Boc-group as usual (30 min.treatment with 4 N HCl in THF). On work-up, a white solid mass wasobtained which on recrystallization gave 4.99 g (72.8%) of the titlecompound: mp 138°- 140°; [α]_(D) ²⁵ =-23.86° (c 1, MeOH).

Anal. Calcd for C₁₇ H₂₀ NO₃ Cl (321.80); C, 63.45; H, 6.26; N, 4.35.Found: C, 63.30; H, 6.02; N, 4.15.

EXAMPLE 21 Ac-Ser(Bzl)-OBzl

HCl.H-Ser(Bzl)-OBzl (3.38 g, 10.5 mmol) was dissolved in 50 ml of DMFand treated with Ac-OSu (1.73 g, 11 mmol) followed by 3 ml of Et₃ N. Themixture was stirred for 18 hours and then acidified with a few ml ofHOAc. Evaporation of the solvent left an oil which was partitionedbetween EtOAc and 5% HOAc. The organic layer was washed with H₂ O (3times), dried over Na₂ SO₄, and evaporated to a clear oil which ontreatment with petroleum ether solidified immediately (3.2 g). Theproduct was recrystallized from EtOAc and petroleum ether to to yield,2.7 g (78.6%); mp 89°-91°; [α]_(D) ²⁵ =-14.91° (c 1, MeOH).

Anal. Calcd for C₁₉ H₂₁ NO₄ (327.38): C, 69.71; H, 6.47; N, 4.28. Found:C, 69.55; H, 6.37; N, 4.37.

The same compound was also prepared by a different route. Thus,H-Ser(Bzl)-OH (18.6 g, 95.4 mmol) was dissolved in 45 ml Triton B,evaporated to dryness, and the residue re-evaporated twice with DMF (100ml each). The residue was then stirred with Ac-OSu (16.9 g, 95.4 mmol)in 150 ml DMF for 20 hours. N-Methylmorpholin was added from time totime in order to maintain the reaction slightly basic. Removal of thesolvent and extraction of the product into EtOAc followed by washingwith small volumes of 10% HOAc, H₂ O (the product is water soluble, usea small volume of H₂ O), dried over Na₂ SO₄, evaporation to drynessagain gave a clear oil (14.5 g, Ac-Ser(Bzl)-OH). The compound failed tocrystallize. It was thus dissolved in a mixture of MeOH and H₂ O (300ml:30 ml), titrated to pH 7.0 with 20% Cs₂ CO₃ and evaporated to a solidmass. The salt was re-evaporated twice more with DMF and stirred withbenzyl bromide (15.4 g, 91 mmol) in 250 ml DMF for 18 hours. Onevaporation of the solvent, the residue was taken up in H₂ O (600 ml)and the oily product formed was extracted into EtOAc. It was washed withH₂ O, dried over Na₂ SO₄ and evaporated to a syrup which on seedingcrystallized immediately. It was recrystallized from EtOAc and petroleumether to yield, 10.42 g (32.2% overall); mp 89°-91°.

EXAMPLE 22 Ac-Ser(Bzl)-HNNH₂

Ac-Ser(Bzl)-OBzl(2.2 g, 6.73 mmol) was dissolved in 75 ml EtOH andstirred gently with 5 ml of H₂ NNH₂ overnight. Some insoluble matterprecipitated was filtered off and the filtrate evaporated to an oilwhich solidified when treated with ether. The product was recrystallizedfrom a small volume of EtOH and ether to yield, 1.40 g (82.8%); mp128°-130°; [α]_(D) ²⁵ =+5.80° (c 1, MeOH).

Anal. Calcd for C₁₂ H₁₇ N₃ O₃ (251.28): C, 57.36; H, 6.82; N, 16.72.Found: C, 57.13; H, 6.87; N, 16.74.

EXAMPLE 23 Boc-Ala-Ala-OH

L-Alanine (3.57 g, 40 mmol) was dissolved in 18.8 ml of Triton B (40mmol), evaporated to dryness, and the oily residue re-evaporated twicewith DMF (30 ml each). The salt obtained was stirred with 11.45 g ofBoc-Ala-OSu (40 mmol) in 40 ml DMF, with 4 ml of NMM added, for 20hours. The solvent was removed and the residue taken up in 10% HOAc (100ml). The product was extracted into EtOAc (4 times 100 ml), washed twicewith a small volume of H₂ O, dried over Na₂ SO₄, evaporated to a smallvolume, and treated with petroleum ether until cloudiness developed. Acrystalline product formed on storage in the refrigerator overnight:Yield, 8.2 g (76.3%); mp 115°-118°.

Anal. Calcd for C₁₁ H₂₀ N₂ O₅.1/2H₂ O (269.30): C, 49.05; H, 7.86; N,10.39. Found: C, 48.90; H, 7.93; N, 10.15.

EXAMPLE 24 HCl.H-Ala-Ala-OH

Boc-Ala-Ala-OH (36.2 g, 139 mmol) was treated with 3 liters of 4 N HClin THF for 30 minutes. Evaporation and work-up as usual gave an oilymass which solidified when treated with ether. The product wasrecrystallized from methanol with ether to yield 9.1 g (33.3%); mp209°-211°.

Anal. Calcd for C₆ H₁₃ N₂ O₃ Cl (196.64): C, 36.65; H, 6.66; N, 14.25.Found: C, 36.85; H, 6.80; N, 14.02.

EXAMPLE 25 Boc-Ala-Ala-OBzl

Boc-Ala-Ala-OH (7.23 g, 27.8 mmol) was dissolved in a mixture of 200 mlMeOH and 20 ml H₂ O.Cs₂ CO₃ solution (20% aqueous) was added until thepH reached 7.0 (˜30 ml) and the resultant neutral solution evaporated todryness. The residue was re-evaporated twice with DMF (150 ml) and thegelatenous solid that remained was stirred in 120 ml DMF with benzylbromide (7.2 g, 42 mmol) for 15 hours. The solvent was then removed byevaporation and the residue treated with 500 ml water. The productprecipitated as an oil which gradually solidified on standing. It wastaken up in EtOAc (400 ml), washed with H₂ O (3 times), dried (Na₂ SO₄),and evaporated to a syrup which on treatment with petroleum ether beganto crystallize. The product was recrystallized from EtOAc and petroleumether to yield, 7.2 g (73.8%); mp 71°-73° (lit. mp 73°-74°; D. A. Lauferet al., J. Amer. Chem. Soc., 90, 2696 (1968)).

EXAMPLE 26 HCl.H-Ala-Ala-OBzl

Boc-Ala-Ala-OBzl (6.0 g, 17.15 mmol) was treated with 380 ml of freshlyprepared 4 N HCl in THF for 30 minutes. The excess acid and solvent wereevaporated off and the remaining syrup re-evaporated twice with freshTHF. The residue solidified immediately when treated with ether. It wasrecrystallized from MeOH and ether to yield, 4.30 g (87.4%); mp154°-156°; [α]_(D) ²⁵ =-38.86° (c 1, MeOH).

Anal. Calcd for C₁₃ H₁₉ N₂ O₃ Cl (286.76): C, 54.45; H, 6.68; N, 9.77.Found: C, 54.44; H, 6.73; N, 9.98.

EXAMPLE 27 Boc-Asp(OBzl)-Ala-Ala-OH.DCHA

HCl.H-Ala-Ala-OH (2.36 g, 12 mmol) was dissolved in 20 ml of DMF,chilled in an ice-bath, and treated with 1.68 ml of Et₃ N (12 mmol)followed by Boc-Asp(OBzl)-OSu (12 mmol). The mixture was stirred gentlyat 0° for 2 hours and then at 25° overnight during which time one moreequivalent of Et₃ N (12 mmol) was added, in small proportions,maintaining the reaction pH near 8.0. A few ml of HOAc was added and theacidified mixture evaporated to dryness. The product formed wasextracted into EtOAc, washed with H₂ O (3 times), dried over Na₂ SO₄,and evaporated to an oily residue (6 g). It was dissolved in EtOAC andtitrated to pH 8.0 with DCHA. The crystalline salt precipitated and wasrecrystallized from i-PrOH and petroleum ether to yield, 5.1 g (65.7%);mp 138°-141°; [α]_(D) ²⁵ =-13.33° (c 1, MeOH).

Anal. Calcd for C₃₄ H₅₄ N₄ O₈ (646.80): C, 63.14; H, 8.42; N, 8.66.Found: C, 63.00; H, 8.28; N, 8.76.

EXAMPLE 28 Ac-Ser(Bzl)-Asp(OBzl)-Ala-Ala-OH

Boc-Asp(OBzl)-Ala-Ala-OH.DCHA (3.5 g, 5.4 mmol) was partitioned between500 ml EtOAc and 350 ml H₂ O containing 10 ml of 10% H₂ SO₄. The aqueouslayer was extracted once more with EtOAc (250 ml) and the combined EtOAclayer washed twice with H₂ O, dried over Na₂ SO₄, and evaporated todryness, leaving a glassy solid of Boc-Asp(OBzl)-Ala-Ala-OH (2.5 g).This material was treated with 200 ml of freshly prepared 4 N HCl in THFfor 30 minutes, evaporated at 32° to a syrup, and re-evaporated twicemore with THF. The oily residue solidified when treated with ether. Thishydrochloride salt of H-Asp(OBzl)-Ala-Ala-OH (1.93 g, 4.83 mmol) wasthen used in the next reaction involving azide coupling withAc-Ser(Bzl)-N₃ that was prepared from 1.24 g Ac-Ser(Bzl)-HNNH₂ (4.9mmol) in 25 ml DMF (-25°) with 7.42 ml of 3.3 N HCl in THF (24.5 mmol)and 0.99 ml of i-amylnitrite (7.35 mmol) stirred at -30° for 30 minutes.The azide solution prepared was cooled down to -35°, mixed with 4.1 mlof Et₃ N and then treated with the white powder ofHCL.H-Asp(OBzl)-Ala-Ala-OH (1.93 g) prepared above. The mixture wasstirred at -20° for 30 minutes and then at 4° for 2 days. Some more Et₃N was added to keep the reaction slightly basic. Work-up as usual gave acrystalline mass which was recrystallized from THF and petroleum etherto yield, 1.85 g (65.6%); mp 167°-170°; [α]_(D) ²⁵ =-18.91° (c 1, DMSO).

Anal. Calcd for C₂₉ H₃₆ N₄ O₉ (584.61): C, 59.58; H, 6.21; N, 9.58.Found: C, 59.35; H, 6.09; N, 9.59.

EXAMPLE 29 Ac-Ser(Bzl)-Asp(OBzl)-Ala-Ala-HNNH₂

Ac-Ser(Bzl)-Asp(OBzl)-Ala-Ala-OH (0.825 g, 1.41 mmol) was dissolved in 4ml DMF and chilled to 0° in an ice-bath. To the solution, H₂ NNH₂ (54.3mg; 1.69 mmol) was added followed by HOBT.H₂ O (0.475 g, 3.10 mmol) andDCC (0.32 g, 1.55 mmol). The mixture was adjusted to pH 7.5 (wet pHpaper) with NMM and stirred at 0° for 2 hours followed by 17 hours at25°. The reaction became a gel during this time. It was diluted withMeOH and the solid material remaining was collected on a suction filterand washed thoroughly with MeOH, ether and petroleum ether to give amaterial melting at 229°-232°. The product was then precipitated fromDMF and MeOH to yield, 0.51 g (61.0%); mp 230°-232°; [α]_(D) ²⁵ =-17.94°(c 1, DMSO).

Anal. Calcd for C₂₉ H₃₈ N₆ O₈ (598.66): C, 58.18; H, 6.40; N, 14.04.Found: C, 58.01; H, 6.38; N, 14.17.

EXAMPLE 30 Boc-Ser(Bzl)-Asp(OBzl)-Ala-Ala-HNNH₂

Boc-Ser(Bzl)-Asp(OBzl)-Ala-Ala-OH (0.55 g, 0.86 mmol) in DMF (4 ml) at0° was treated with H₂ NNH₂ (32.9 mg, 1.03 mmol), HOBT.H₂ O (288.2 mg,1.88 mmol), and DCC (193.9 mg, 0.941 mmol). The mixture was brought topH 7.5 with a few drops of NMM and then stirred at 0° for 2 hoursfollowed by 25° for 17 hours. The solid by-products formed were filteredoff and the filtrate evaporated to dryness. The oily residue wascrystallized from i-PrOH twice: Yield, 0.13 g (23.1%); mp 184°-187°;[α]_(D) ²⁵ =-14.22° (c 1, DMSO).

Anal. Calcd for C₃₂ H₄₄ N₆ O₉ (656.74); C, 58.52; H, 6.75; N, 12.80.Found: C, 58.55; H, 6.68; N, 12.68.

EXAMPLE 31 Boc-Asn-Ala-Ala-OBzl

HCl.H-Ala-Ala-OBzl (4.25 g, 14.83 mmol) was dissolved in DMF (60 ml),cooled to 0°, and stirred with Boc-Asn-OH (3.45 g, 14.83 mmol), HOBT(4.02 g, 29.6 mmol), NMM (2 ml) and DCC (3.37 g, 16.35 mmol) at 0° for 2hours and then at 25° for 20 hours. More NMM was added from time to timein order to maintain the reaction slightly basic. The insolubleby-products formed was filtered off and the filtrate evaporated todryness. The oily residue solidified immediately when treated withwater. It was taken up in EtOAc, washed with H₂ O (3 times), dried (Na₂SO₄), and evaporated to a smaller volume when the product began tocrystallize. Equal volumes of petroleum ether was added and the mixturewas left to stand overnight. The crude product was recrystallized fromTHF and petroleum ether: yield, 5.2 g (75.6%); mp 153°-155°; [α]_(D) ²⁵=- 55.61 (c 1, MeOH).

Anal. Calcd for C₂₂ H₃₂ N₄ O₇ (464.51): C, 56.89; H, 6.94; N, 12.06.Found: c, 56.66; H, 7.08; N, 11.81.

EXAMPLE 32 Ac-Ser(Bzl)-Asn-Ala-Ala-OBzl

Boc-Asn-Ala-Ala-OBzl (2.2 g, 4.75 mmol) was treated with 220 ml of 4.0 NHCl in THF for 30 minutes. The excess acid and solvent were evaporatedoff and the residue re-evaporated twice with fresh THF. The oily productsolidified when treated with ether. It was triturated with more freshether and collected to give a white powder (1.85 g). The hydrochloridesalt thus obtained was dissolved in 40 ml DMF and chilled to 0° whenAc-Ser(Bzl)-OH.DCHA (1.97 g, 4.75 mmol) was added. After stirring at 0°for 30 minutes, precipitation of DCHA.HCl was observed and HOBT (1.19 g)was added followed by DCC (1.08 g, 5.23 mmol). The reaction was thenadjusted to pH 7.5 (wet pH paper) with a few drops of NMM and stirred at0° for 2 hours and then at 25° overnight. The insoluble by-product wasremoved by filtration and the filtrate evaporated to a slightly coloredsolid mass. It was washed thoroughly with H₂ O and EtOAc to give a buffcolored powder which was crystallized from DMF (40 ml) and i-PrOH (500ml): yield, 1.57 g (56.9%); mp 213°-215°; [α]_(D) ²⁵ =-23.11° (c 1,DMSO).

Anal. Calcd for C₂₉ H₃₇ N₅ O₈ (583.63): C, 59.68; H, 6.39; N, 12.00.Found: C, 59.36; H, 6.59; N, 11.97.

EXAMPLE 33 Ac-Ser(Bzl)-Asn-Ala-Ala-HNNH₂

Ac-Ser(Bzl)-Asn-Ala-Ala-OBzl (1.57 g, 2.69 mmol) was dissolved in 20 mlDMF and stirred with 2 ml of H₂ NNH₂ for 18 hours. The solid productformed was collected and washed thoroughly with DMF, EtOH, and ether:yield, 1.22 g (89.6%); mp 262°-264°; [α]_(D) ²⁵ =-26.70° (c 1, DMSO).

Anal. Calcd for C₂₂ H₃₃ N₇ O₇ (507.54): C, 52.06; H, 6.55; N, 19.32.Found: C, 51.48; H, 6.58; N, 19.22.

EXAMPLE 34 Ac-Ser(Bzl)-Asp(OBzl)-Ala-Ala-OBzl

HCl.H-Ala-Ala-OBzl (1.68 g, 5.83 mmol) was dissolved in a mixture of DMF(8 ml) and CH₂ Cl₂ (20 ml) then cooled in an ice-bath. NMM (0.6 ml),Boc-Asp(OBzl)-OH (1.88 g, 5.83 mmol), and DCC (1.27 g, 6.14 mmol) wereadded in that order and the mixture stirred at 0° for 2 hours and thenat 25° for 15 hours during which time a few more drops of NMM was addedin order to maintain the reaction slightly basic. The insolubleby-products were filtered off and the filtrate evaporated to dryness.The residue was extracted into EtOAc, washed with H₂ O (3 times), driedover Na₂ SO₄, and evaporated to an oil. It was taken up in THF (55 ml),filtered to remove residual dicyclohexylurea, and evaporated to a clearoil (3.3 g). The product Boc-Asp(OBzl)-Ala-Ala-OBzl did not crystallize.It was then treated with 200 ml freshly prepared 4.0 HCl in THF for 30minutes and worked-up as usual to give HCl.H-Asp(OBzl)-Ala-Ala-OBzl(1.89 g, 3.85 mmol) as an amorphous powder. The tripeptide esterhydrochloride was then dissolved in DMF (22 ml), chilled in an ice-bath,and stirred with Ac-Ser(Bzl)-OH.DCHA (1.61 g, 3.85 mmol) for 30 minutesfollowed by addition of HOBT (1.04 g, 7.7 mmol), NMM (0.4 ml), and DCC(0.82 g, 4.24 mmol). The mixture was stirred at 0° for 2 hours and thenat 25° for 17 hours. The insoluble by-products formed were filtered offand the filtrate evaporated to dryness. The solid mass remaining wasdissolved in EtOAc, washed with H₂ O, 5% NaHCO₃, 5% HOAc, H₂ O, anddried over Na₂ SO₄. On evaporation to a smaller volume, a crystallineproduct started to form. The solution was treated with equal volumes of25% petroleum ether in EtOAc and left standing to complete thecrystallization. The product was recrystallized from i-PrOH and pentaneto yield, 0.91 g (23.2%); mp 154°-158°; [α]_(D) ²⁵ =-35.95° (c 1, MeOH).

Anal. Calcd for C₃₆ H₄₂ N₄ O₉ (674.73): C, 64.08; H, 6.27; N, 8.30.Found: C, 63.86; H, 6.24; N, 8.22.

EXAMPLE 35 Ac-Ser-Asp-Ala-Ala-OH

Ac-Ser(Bzl)-Asp(OBzl)-Ala-Ala-OBzl (0.6 g, 0.888 mmol) was hydrogenatedin a Parr apparatus at 50 psi in the presence of 0.62 g catalyst (5% Pdon BaSO₄) in a solvent mixture consisting of 35 ml MeOH, 15 ml H₂ O, and1 ml HOAc for 4 hours. The catalyst was filtered off and the filtrateconcentrated to a smaller volume and lyophilized to give 0.38 g crudematerial. Tlc showed two minor contaminants in addition to the majorcomponent. The compound was then purified on a Bio-Rad AGl×2 column(3×24 cm) which was washed with 0.2 M NaOH, H₂ O, 0.2 M HOAc, andequilibrated with pH 8.5 buffer of 0.025 M ammonium acetate. The samplewas applied at this pH and the column eluted with 250 ml each of thefollowing acetic acid solutions in the order of: 0.25 M, 0.5 M, 1.0 M,1.5 M, 2.0 M, 2.5 M, 3.0 M, 3.5 M, 4.0 M, and 4.5 M. The flow rate wasadjusted to approximately 50 ml/hr and each 10 ml fraction was collectedinto an LKB fraction collector. The compound was located by spotting a 5μl portion from each tube on silica get tlc plate (Merck 60, F-254) anddeveloped in the solvent system of n-BuOH:HOAc:EtOAc:H₂ O (1:1:1:1) andthen stained with chlorine-tolidine reaction. The fractions containingthe major component were pooled and lyophilized twice to give 310 mg(86.3%) of the desired product; [α]_(D) ²⁵ =-77.01° (c 1, 10% HOAc).

Anal. Calcd for C₁₅ H₂₄ N₄ O₉ (404.37): C, 44.55; H, 5.98; N, 13.86.Found: C, 44.25; H, 5.98; N, 13.76.

EXAMPLE 36 Ac-Ser-Asn-Ala-Ala-OH

Ac-Ser(Bzl)-Asn-Ala-Ala-OBzl (0.58 g, 0.992 mmol) was dissolved in asolvent mixture of MeOH (35 ml), DMF (5 ml), H₂ O (15 ml) and HOAc (1.5ml) and then hydrogenated as described above at 50 psi for 5 hours inthe presence of 1.5 g catalyst. The catalyst was filtered off and thefiltrate evaporated to a smaller volume and lyophilized to give 0.411 gof crude product. It was purified on the same column with the sameprocess as described for Ac-Ser-Asp-Ala-Ala-OH (BioRad AGl×2ion-exchange). The main fraction was pooled and lyophilized: yield, 288mg (72.2%); [α]_(D) ²⁵ =-83.61° (c 1, 10% HOAc).

Anal. Calcd for C₁₅ H₂₅ N₅ O₈ (403.39): C, 44.66; H, 6.25; N, 17.36.Found: C, 44.42; H, 6.35; N, 17.16.

EXAMPLE 37 Boc-Ser(Bzl)-Glu(OBzl)-OH.CHA

H-Glu(OBzl)-OH (39.4 g, 166 mmol) was stirred with Boc-Ser(Bzl)-OSu(65.0 g, 166 mmol) in 900 ml DMF overnight in the presence of Et₃ N (2.3ml, 165 mmol). More Et₃ N was added during this time in order tomaintain the reaction slightly basic. The clear solution was evaporatedto dryness and the oily residue partitioned between EtOAc (1.5 liters)and 5% HOAc (2 liters). The organic layer was washed with H₂ O (2times), dried over Na₂ SO₄, and concentrated to a clear oil (90.0 g)which was taken up in 3 liters of ether and treated with 25 ml ofcyclohexylamine The solid formed was recrystallized from MeOH and ether:yield, 76.2 (74.8%); mp 154°-156.5°; [α]_(D) ²⁵ =+6.32° (c 1, MeOH).

Anal. Calcd for C₃₃ H₄₇ N₃ O₈ (613.73): C, 64.58; H, 7.72; N, 6.85.Found: C, 64.78; H, 7.90; N, 6.80.

EXAMPLE 38 Boc-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH

Boc-Ser(Bzl)-Glu(OBzl)-OH.CHA (76.2 g, 124 mmol) was suspended in amixture of 1.5 liters each of H₂ O and EtOAc. To this mixture, 10% H₂SO₄ was added until it became acidic (˜pH 2.5) and the solid dissolved.The organic layer containing the dipeptide free acid was washed with H₂O (2 times), dried, and evaporated to dryness leaving a clear oil (68.5g). It was treated with 3 liters of freshly prepared 4.1 N HCl in THFfor 45 minutes and evaporated to an oily residue which was re-evaporatedtwice more with THF. The residue (HCl.H-Ser(Bzl)-Glu(OBzl)-OH) wasdissolved in DMF (500 ml), chilled to 0°, and treated withBoc-Ser(Bzl)-OSu (48.66 g, 124 mmol), followed immediately by 27 ml ofEt₃ N. The mixture was stirred overnight at 25° during which time moreEt₃ N was added occassionally in order to maintain the reaction slightlybasic. Some small quantities of insoluble matters were removed byfiltration and the filtrate evaporated to an oil which was taken up inEtOAc, washed with 5% HOAc (acidify with more HOAc if necessary), H₂ O,dried over Na₂ SO₄, and evaporated again to dryness. The product wascrystallized from EtOAc with petroleum ether: yield, 71.8 g (83.7%); mp112°-113°; [α]_(D) ²⁵ =+17.91° (c 1, THF).

Anal. Calcd for C₃₇ H₄₅ N₃ O₁₀ (691.75): C, 64.24; H, 6.56; N, 6.07.Found: C, 64.32; H, 6.30; N, 6.19.

EXAMPLE 39 Boc-Thr(Bzl)-OSu

Boc-Thr(Bzl)-OH (23.2 g, 75 mmol) was stirred with HOSu (9.5 g, 82.5mmol) and DCC (17.0 g, 82.5 mmol) for 3 hours. The insoluble by-productwas filtered off and the filtrate evaporated to an oil which was takenup in ether (400 ml). On standing at 4° for a few hours more by-product(DCU) was formed. It was filtered off again and the filtrate evaporatedto a semisolid which was crystallized from i-PrOH, to give 29.5 g (mp70°-75°) of crude material. The crude was recrystallized from EtOH;yield, 21.2 g (69.8%); mp 104°-106°; [α]_(D) ²⁵ =+6.04° (c 1, CHCl₃);[lit. mp 94°; [α]_(D) ²² =-8.5° (c 1, MeOH); Danho and Li, Int. J.Peptide Protein Res., 3, 81 (1971)].

Anal. Calcd for C₂₀ H₂₆ N₂ O₇ (406.44): C, 59.10; H, 6.44; N, 6.89.Found: C, 59.16; H, 6.52; N, 6.68.

EXAMPLE 40 Boc-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH

Boc-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH (71.6 g, 104 mmol) was treated with2.7 liters of freshly prepared 3.9 N HCl in THF for 45 minutes. Themixture was evaporated to dryness and the residue evaporated twice morewith THF to give a solid mass (59.3 g; mp 161°-165°). It was collectedand washed with ether and stirred in 500 ml of DMF with Boc-Thr(Bzl)-OSu(38.2 g, 94 mmol) in the presence of Et₃ N (25 ml) at 0° for 1 hour andthen at 25° for 15 hours. More Et₃ N (14.5 ml) was added in serveralportions during this time to maintain the reaction slightly basic. Someinsoluble matter formed was filtered off and the filtrate evaporated toan oil which was dissolved in EtOAc (1.5 liters), washed with 5% HOAc,H₂ O (2 times), dried (Na₂ SO₄), and evaporated to a solid mass. Theproduct was recrystallized from EtOAc and petroleum ether to yield, 64.8g (78.1%); mp 115°-118°; [α]_(D) ²⁵ =+11.64° (c 1, DMSO).

Anal. Calcd for C₄₈ H₅₈ N₄ O₁₂ (882.97): C, 65.29; H, 6.62; N, 6.35.Found: C, 64.99; H, 6.77; N, 6.32.

EXAMPLE 41 Boc-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH

Boc-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH (54.5 g, 61.7 mmol) wastreated with HCl in THF (1.5 liters; 4.1 N; 40 min) and worked up asusual to give HCl.H-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH (46.3 g,56.6 mmol) as a white powder. It was then stirred in DMF (500 ml) withBoc-Asp(OBzl)-OSu (23.7 g, 56 mmol) at 0° for 2 hours in the presence ofEt₃ N (16 ml). The mixture was further stirred at 25° for 15 hoursduring which time 7.4 ml more of Et₃ N was added. The product was workedup as usual and crystallized from CH₂ Cl₂ and petroleum ether: yield,50.35 g (82.7%); mp 111°-113°; [α]_(D) ²⁵ =+7.21° (c 1, DMSO).

Anal. Calcd for C₅₉ H₆₉ N₅ O₁₅ (1088.22): C, 65.12; H, 6.39; N, 6.44.Found: C, 65.06; H, 6.27; N, 6.58.

EXAMPLE 42 Boc-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH

Boc-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH (50.0 g, 46 mmol)was deprotected with HCl (4.15 N) in THF and worked up as usual to give45.4 g of white solid. It was dissolved in THF (1.5 liters) and treatedwith ether (7 liters). On standing at 0° overnight a white solid powderwas obtained (44.0 g, mp 179°-184°). Part of this material,HCl.H-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH (43.7 g, 42.7mmol) was then dissolved in 500 ml of DMF, cooled to 0°, and treatedwith Boc-Val-OSu (15.4 g, 49 mmol) and Et₃ N (10 ml). The mixture wasstirred for 15 hours during which time more Et₃ N (7.9 ml) was added inseveral portions maintaining the reaction slightly basic. The insolublematter was removed by filtration and the filtrate evaporated to dryness.The oily residue was dissolved in CH₂ Cl₂, washed with 5% HOAc, H₂ O,dried (Na₂ SO₄), and evaporated to a smaller volume (0.5 liter) whentreated with petroleum ether. The product crystallized slowly duringovernight standing. It was recrystallized from THF and i-PrOH to yield,26.3 g (51.4%); mp 174°-177°; α]_(D) ²⁵ =+0.84° (c, 1, THF).

Anal. Calcd for C₆₄ H₇₈ N₆ O₁₆ (1187.31): C, 64.74; H, 6.62; N, 7.08.Found: C, 64.60; H, 6.71; N, 6.91.

EXAMPLE 43 Boc-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-HNNH₂

Boc-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-OH (13.0 g; 10.94mmol) was dissolved in DMF (50 ml), cooled to 0°, and treated with H₂NNH₂ (0.421 g; 13.14 mmol), HOBT (3.688 g; 24.1 mmol), and DCC (2.48 g;12.04 mmol). NMM was then added until the reaction showed pH 7.5 (wet pHpaper). The mixture was stirred for 18 hours and filtered to remove theinsoluble by-products. The filtrate was evaporated to dryness and theresidue treated with H₂ O. The solid formed was collected andcrystallized from DMF and i-PrOH: yield, 8.7 g (66.4%); mp 215°-218°;[α]_(D) ²⁵ =+7.62° (c 1, DMSO).

Anal. Calcd for C₆₄ H₈₀ N₈ O₁₅ (1201.38): C, 63.98; H, 6.71; N, 9.33.Found: C 64.07; H, 6.85; N, 9.17.

EXAMPLE 44 Boc-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH

Boc-Thr(Bzl)-Lys(Z)-OH (14.0 g, 24.5 mmol) was treated with 500 ml of4.0 N HCl in THF for 30 minutes, evaporated to dryness, andre-evaporated twice with fresh THF. The oily residue solidified whentreated with ether. The dried powder (11.4 g, 21.6 mmol) of thedipeptide hydrochloride salt was then dissolved in 140 ml DMF, cooled to0°, and treated with Boc-Thr(Bzl)-OSu (8.8 g, 21.6 mmol) followed by 3.5ml of Et₃ N. A few drops of Et₃ N was added to maintain a slightly basiccondition while the mixture was stirred for an additional 24 hours at25°. It was acidified with 5 ml of HOAc and then diluted with a largevolume of water. The solid crude product precipitated was collected,dissolved in EtOAc, washed with H₂ O, dried over Na₂ SO₄, and evaporatedto dryness, leaving a glassy solid mass. Crystallized from EtOAc andpetroleum ether: yield, 13.8 g (83.7%); mp 110°- 112°; [α]_(D) ²⁵=+19.45° (c 1, EtOAc).

Anal. Calcd for C₄₁ H₅₄ N₄ O₁₀ (762.87): C, 64.55; H, 7.13; N, 7.34.Found: C, 64.41; H, 7.09; N, 7.44.

EXAMPLE 45 Boc-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH

Boc-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH (41.5 g, 54 mmol) was treated with 500ml of freshly prepared 3.55 n HCl in THF for 25 minutes and evaporatedto a syrup which was re-evaporated twice with fresh THF. The oilyresidue soldified when treated with ether. It was collected and washedwith ether to give 37.4 g of crude hydrochloride salt of the tripeptide,dissolved in 500 ml of DMF, chilled to 0°, and treated with Boc-Ile-OSu(17.4 g, 53 mmol) followed by 16 ml of Et₃ N. The mixture was stirred at25° overnight during which time more Et₃ N was added in small portions(6.2 ml total) to maintain a slightly basic condition. The resultantmixture was filtered and the filtrate evaporated to an oil which wasextracted into EtOAc, washed with 5% HOAc, H₂ O dried over Na₂ SO₄, andevaporated to give a yellowish oil. It was crystallized from EtOAc andpetroleum ether. The crude solid thus obtained (39.6 g, mp 140°-142°)was found to be contaminated with several minor impurities. The materialwas then chromatographed on a silica gel 60 (70-230 mesh) column (4.7×67cm) using CHCl₃ -MeOH (95:5) as eluent. The fractions containing thedesired product (monitored by tlc) were pooled and evaporated to give anoily product which was crystallized from CHCl₃ and petroleum ether:yield, 19.1 g (41.2%); mp 144°-146°; [α]_(D) ²⁵ =+2.40° (c 1, CHCl₃).

Anal. Calcd for C₄₇ H₆₅ N₅ O₁₁ (876.06): C, 64.44; H, 7.48; N, 7.99.Found: C, 63.47; H, 7.38; N, 7.80.

EXAMPLE 46Boc-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH

Boc-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH (0.439 g, 0.496 mmol) was treatedwith 4 N HCl in THF for 30 minutes and worked up as usual to give 0.39 gof HCl.H-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH.Boc-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-HNNH₂ (0.59 g,0.492 mmol) was then dissolved in 6 ml of DMF, cooled to -25°, andtreated with 0.57 ml of 4.3 N HCl in THF (2.46 mmol) followedimmediately by 0.1 ml of i-amylnitrite (0.74 mmol). After stirring at-20--25° for 30 minutes, the temperature was lowered to -35° when 0.42ml of Et₃ N and HCl.H-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH (0.39 g) preparedabove were added. The mixture was stirred at -20° for 30 minutes andthen at 4° for 48 hours during which time Et₃ N was added from time totime in order to keep the pH at about 7.5. The reaction was then dilutedwith 250 ml of 5% HOAc and the solid product formed was collected andwashed with H₂ O, MeOH, ether, and dried over NaOH pellets in vacuo togive 0.82 g of crude material (mp 244°-254°). It was dissolved in DMSOand precipitated by addition of MeOH: yield, 0.698 g (81.7%); mp268°-271°.

Anal. Calcd for C₁₀₆ H₁₃₃ N₁₁ O₂₄ (1945.21): C, 65.46; H, 6.89; N, 7.92.Found: C, 65.32; H, 6.99; N, 8.18.

EXAMPLE 47Ac-Ser(Bzl)-Asn-Ala-Ala-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH

Boc-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH(0.698 g, 0.358 mmol) was treated with 10 ml of TFA for 30 minutes andthe peptide salt was precipitated with ether. It was collected on asuction filter, washed with ether and dried to give 0.652 g of material(0.333 mmol as TFA salt). In a separate flask,Ac-Ser(Bzl)-Asn-Ala-Ala-HNNH₂ (0.17 g, 0.335 mmol) was suspended in 7 mlDMF and treated with 0.27 ml of 6.18 N HCl in THF at -20°. To themixture, 0.68 ml of 10% i-amylnitrite in DMF was added and the solutionstirred at the same temperature for 30 minutes. The temperature waslowered to -30° when 0.2344 ml of Et₃ N (1.675 mmol) was added followedby the TFA salt of decapeptide (0.652 g) prepared above. The mixture wasdiluted with 3 ml of DMSO at -20° and adjusted to a slightly basiccondition (pH 7.5 to wet pH paper) with a few drops of Et₃ N. It wasstirred at -20° for 30 minutes and then at 4° for 5 days. More DMSO (5ml) and Et₃ N were added during this period of time to maintain theslightly basic conditions and to keep the reaction from becoming a gel.The entire solution was then poured into 5% HOAc (250 ml) to give awhite precipitate which was collected, washed with H₂ O, MeOH, ether,and dried. The crude product (0.702 g; mp 290°-291°) was reprecipitatedfrom DMSO with MeOH: yield, 0.348 g (42.0%); mp 296°-298° (d); [α]_(D)²⁵ -+3.77° (c 1, DMSO).

Anal. Calcd for C₁₂₃ H₁₅₃ N₁₆ O₂₉ (2319.58): C, 63.68; H, 6.65; N, 9.66.Found: C, 63.44; H, 6.83; N, 9.50.

EXAMPLE 48Ac-Ser(Bzl)-Asp(OBzl)-Ala-Ala-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH

Ac-Ser(Bzl)-Asp(OBzl)-Ala-Ala-HNNH₂ (0.408 g, 0.68 mmol) suspended in 10ml DMF was cooled to -20° and treated with freshly prepared 5.43 N HClin THF (0.627 ml, 3.4 mmol) followed by 10% i-amylnitrite in DMF (1.39ml, 1.03 mmol). After stirring for 30 minutes, it was cooled down to-30° when Et₃ N (0.476 ml, 3.4 mmol) was added followed by the TFA saltof the decapeptideH-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OH(1.334 g, 0.68 mmol). The mixture was stirred at -20° for 30 minutes andthen at 4° for 5 days during which time more Et₃ N and DMSO were addedin order to maintain the reaction at a slightly basic condition and tokeep the gel from forming. The entire reaction was poured into 5% HOAc(300 ml) and the solid precipitate formed was collected, washed with H₂O, MeOH, ether, and dried to give 1.49 g of material melting at296°-299°. The product was reprecipitated from DMSO with MeOH: yield,1.40 g (85.37%); mp 297°-299°; [α]_(D) ²⁵ =+6.37° (c 1, DMSO).

Anal. Calcd for C₁₃₀ H₁₅₈ N₁₅ O₃₀ (2410.684): C, 64.77; H, 6.61; N,8.72. Found: C, 64.53; H, 6.73; N, 8.81.

EXAMPLE 49Ac-Ser(Bzl)-Asp(OBzl)-Ala-Ala-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Thr(Bzl)-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-Asp(OBzl)-Leu-Lys(Z)-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl

The tetradecapeptide of Example 48 (1.35 g, 0.558 mmol) was stirred withHOBT. H₂ O (0.188 g, 1.23 mmol) for a few minutes in a mixture of 15 mleach of DMF and DMSO. The mixture was then cooled in an icebath when DCC(0.126 g, 0.614 mmol) was added and the stirring continued for 24 hoursat the same temperature. In a separate flaskBoc-Asp(OBzl)-Leu-Lys(Z)-Glu(OBzl)-Lys(Z)-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl (4.0 g, 1.43 mmol) was treated with 40 mlof TFA for 25 min. and the TFA salt of the ensuing tetradecapeptideprecipitated quickly with addition of a large volume of ether. The solidformed was collected and washed thoroughly with ether to give 3.74 g ofwhite powder (TFA salt of C-terminal tetradecapeptide). Part of thismaterial (1.567 g, 0.5583 mmol) was added to the active ester derivedfrom the amino terminal tetradecapeptide as prepared above in a DMF-DMSOmixture. A few drops of NMM was added to bring the pH of the reaction to7.5-8.0, and the stirring was continued for 1 hour at 0° and then 5 daysat 25°. The reaction was then poured into 1.51 of 5% acetic acid. Theprecipitated product was washed thoroughly with H₂ O, MeOH, DMF, MeOHand ether to give 2.21 g of the desired product melting above 300°.

Anal. Calcd for C₂₇₃ H₃₃₅ N₃₃ O₆₃ (5089.69): C, 64.47; H, 6.64; N, 9.09Found: C, 63.27; H, 6.50; N, 8.88.

Amino Acid Anal.: Asp, 3.83; Thr, 3.12; Ser, 2.81; Glu, 6.14; Ala, 3.00;Val, 2.89; Ile, 1.30; Leu, 0.77; Lys, 3.72; NH₃, 2.01.

EXAMPLE 50Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn[Thymosin α₁ ].

The product of Example 49 (2.21 g, 0.435 mmol) was dissolved in 8 ml ofTFA, mixed with 4 ml of anisole and stirred with anhydrous HF at 0° for30 minutes. The acids were removed at 0° (vacuum distillation) and thesolid residue which remained was dissolved in 200 ml H₂ O, washed twicewith ether, evaporated to half of the volume and lyophilyzed to give 1.1g of crude product. It was purified by passing through a Sephadex G-10column (3×80 cm; 0.2 M HOAc) and then a DEAE-Sephadex column (3×75 cm)eluted with increasing concentrations of ammonium acetate (pH 7.0, 0.025M 0.25 M) followed by dilute acetic acid. The fractions containing thedesired material were pooled and lyophilized to give 0.281 g ofamorphous white powder.

On acrinamide gel isolectrofocusing, the syntheticmaterial migratesidentically as the natural thymosin α₁ isolated from bovine thymus gland(A.L. Goldstein, T.L.K. Low, C.Y. Lai and S.S. Wang, U.S. PatentApplication Serial No. 766,638, Feb. 8, 1977; A.L. Goldstein et al.,Proc. Natl. Acad. Sci. (U.S.A.), 74, 725 (1977)).

Amino Acid Anal.: Asp, 3.86; Thr, 3.22; Ser, 3.05; Glu, 6.[3; Ala, 3.]5;Val, 3.04; Ile, 1.12; Leu, 0.94; Lys, 3.88; NH₃, 2.25.

EXAMPLE 51Ac-Ser(Bzl)-Asn-Ala-Ala-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-Asp(OBzl)-Leu-Lys(Z)-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl.

The product of Example 47 (0.866 g, 0.373 mmol) was stirred with HOBT .H₂ O (0.126 g, 0.82 mmol) in a mixture of DMSO (8 ml) and DMF (6 ml) andchilled in an icebath. The mixture was treated with DCC (0.085 g, 0.411mmol) and then stirred at 0° for 24 hours. It was then mixed with theTFA salt of the C-terminal tetradecapeptide prepared as in Example 49(1.05 g, 0.373 mmol) and 2 ml more of DMSO. A few drops of NMM wereadded to bring the pH to 7.5-8.0 and the stirring was continued at 0°for 1 hour and then at 25° for 5 days. Worked up as mentioned above inExample 49 gave 1.5775 g of fully protected octacosapeptide.

Anal. Calcd. for C₂₆₆ H₃₃₀ N₃₄ O₆₂ (4995.51): C, 63.95; H, 6.66; N, 9.53Found: C, 63.20; H, 6.60; N, 9.49.

Amino Acid Anal. Asp, 3.96; Thr, 2.87; Ser, 2.46; Glu, 6.03; Ala, 3.05;Val, 3.00; Ile, 1.04; Leu, 0.76; Lys, 3.91; NH₃, 4.45.

EXAMPLE 52Ac-Ser-Asn-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Blu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn [(Asn²)-Thymosin α₁ ].

The compound of Example 51 (1.5775 g, 0.3157 mmol) was dissolved in 10ml of TFA which contained 3 ml of anisole. The mixture was stirred with45 ml of HF at 0° for 30 min and then worked up as described for Example50. Purification on Sephadex G-10 and DEAE-sephadex columns as describedabove gave 0.283 g of white amorphous product. The compound migrated atthe position slightly less acidic than natural thymosin α₁ on acrinamidegel isoelectrofocusing in agreement with difference in the structure.

Amino Acid Anal. Asp, 3.89; Thr, 3.00; Ser, 2.93; Glu, 5.68; Ala, 3.02;Val, 2.63; Ile, 1.12; Leu, 0.86; Lys, 4.03.

EXAMPLE 53 Boc-Glu(OBzl)-Lys(Z)-Lys(Z)-HNNH₂

Boc-Glu(OBzl)-Lys(Z)-Lys(Z)-OH (43.2 g, 50 mmol) was treated in DMF (280ml) at 0° with H₂ NNH₂ (1.76 ml, 55 mmol) and HOBT . H₂ O (15.3 g, 100mmol) followed by DCC (11.33 g, 55 mmol). The mixture was stirred at 0°for 2 hours and then at 25° overnight during which time some NMM wasadded to maintain the reaction slightly basic. On evaporation of thesolvents, the oily residue remained was treated with a large volume ofwater which turned the crude product into a solid mass. It was collectedwas washed with H₂ O, i-PrOH and ether. The powder obtained wascrystallized twice from i-PrOH: yield, 31.93 g (73.0%); mp 164°-169°;[α]_(D) ²⁵ =1.37° (c 1, THF).

Anal. Calcd for C₄₅ H₆₁ N₇ O₁₁ (875.99): C, 61.69; H, 7.02; N, 11.19.Found: C, 61.86; H, 7.06; N, 10.93.

EXAMPLE 54 Boc-Asp(OBzl)-Leu-Lys(Z)-HNNH₂

Boc-Asp(OBzl)-Leu-Lys(Z)-OH (14.8 g, 21.0 mmol) was treated in DMF (80ml) at 0° with H₂ NNH₂ (0.80 ml, 25.0 mmol), HOBT . H₂ O (7.1 g, 46.2mmol), and DCC (4.7 g, 23.0 mmol). The mixture was stirred at 0° (2hours) and then at 25° (17 hours). Work-up as the above compound gave9.2 g (61.5%) of the desired product: mp 133°-137°; [α]_(D) ²⁵ =-19.36°(c 1, THF).

Anal. Calcd for C₃₆ H₅₂ N₆ O₉ (712.82): C, 60.65; H, 7.35; N, 11.79.Found: C, 60.80; H, 7.42; N, 11.51.

EXAMPLE 55 Nα-Boc-α-benzyl-L-aspartyl benzhydrylamine resin.

Benzhydrylamine resin (5 g, Beckman Lot No. 00658; N, 0.52%, 0.371mmol/g) was stirred with 125 ml of 25% Et₃ N in CH₂ Cl₂ for 10 min andthen washed a few times with fresh CH₂ Cl₂. It was then suspended in 120ml of CH₂ Cl₂ and stirred with Nα-Boc-α-benzyl-L-aspartic acid (1.62 g,5.0 m mol) and DCC (1.03 g, 5.0 m mol) for 17 hr. The amino acylatedresin was collected, washed thoroughly with CH₂ Cl₂, DMF and MeOH. Afterdrying, the resin was benzoylated with 1.5 ml pyridine and 1.65 mlbenzoyl chloride in 55 ml of CH₂ Cl₂ for 15 min at 0°. The resin waswashed with CH₂ Cl₂, MeOH to give 5.35 g of desired material. Amino acidanalysis indicated that there was 0.296 m mol/g of aspartic acid.

EXAMPLE 56Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn(Thymosin α₁).

Nα-Boc-α-benzyl-L-aspartyl benzhydrylamine resin (2.5 g, 0.74 m mol) wasplaced in the reaction vessel of an automatic solid phase peptidesynthesis apparatus which was programmed to perform the following steps(40 ml solvent or reagents, unless otherwise stated) in each cycle:

(1) three washings with CH₂ Cl₂,

(2) prewash with 33% TFA in CH₂ Cl₂,

(3) stir 25 min with 33% TFA in CH₂ Cl₂,

(4) three washings with CH₂ Cl₂,

(5) prewash with 10% Et₃ N in CH₂ Cl₂,

(6) stir 10 min with 10% Et₃ N in CH₂ Cl₂,

(7) three washings each with CH₂ Cl₂, i-PrOH, CH₂ Cl₂,

(8) stir 120 min with 2.22 m mol each of Boc-Glu(OBzl)-OH and DCC in CH₂Cl₂,

(9) three washings each with CH₂ Cl₂, DMF, i-PrOH, CH₂ Cl₂,

(10) stir 10 min with 10% Et₃ N,

(11) three washings with CH₂ Cl₂,

(12) stir 120 min with 1.11 m mol each of Boc-Glu(OBzl)-OH and DCC inCH₂ Cl₂,

(13) three washings each with CH₂ Cl₂, DMF, i-PrOH.

The synthetic cycle was repeated using the following amino acids,sequentially, one at a time in step 8 and step 12:

Boc-Ala-OH, Boc-Glu(OBzl)-OH, Boc-Glu(OBzl)-OH, Boc-Val-OH, Boc-Val-OH,

Boc-Glu(OBzl)-OH, Boc-Lys(Cl₂ Z)-OH, Boc-Lys(Cl₂ Z)-OH,Boc-Glu(OBzl)-OH,

Boc-Lys(Cl₂ Z)-OH, Boc-Leu-OH, Boc-Asp(OBzl)-OH, Boc-Lys(Cl₂ Z)-OH,Boc-Thr

(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Ile-OH, Boc-Glu(OBzl)-OH,Boc-Ser(Bzl)-OH,

Boc-Ser(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Asp(OBzl)-OH, Boc-Val-OH,Boc-Ala-OH,

Boc-Ala-OH, Boc-Asp(OBzl)-OH, Boc-Ser(Bzl)-OH, and CH₃ COOH.

On completion of the automated synthetic cycles, 4.15 g of protectedpeptide resin was obtained. Part of this material (0.91 g) was thentreated with 10 ml of anhydrous HF in the presence of 1 ml of anisole at0° for 60 min. After evaporation of the acid (in vacuo, 0° ), theresidue was extracted with 5 ml of TFA, diluted with water, washedseveral times with ether, evaporated to smaller volume and desalted on aSephadex G-10 column (2.5×60 cm). The fractions containing the material(absorption at 215 mm) were pooled and lyophilized to give 127 mg ofcrude product. It was then chromatographed on a DEAE-Sephadex A-25column (1.5×70 cm) using a linear gradient made up from 1 L each of 0.05m Tris-Hcl (pH8.0) with and without 0.3 M NaCl. The main peak waspooled, desalted on the Sephadex G-10 column (0.2M HOAc) and lyophilizedto give 34 mg of product migrating identically with the natural thymosinα₁ and also with the synthetic thymosin α₁ prepared by the conventionalprocess. The overall yield is calculated to be 6.74% based on theaspartic acid content of the Nα-Boc-α-benzyl-L-aspartyl benzhydrylamineresin. Amino acid composition of the product is Asp, 4.10; Thr, 2.78;Ser, 3.22; Glu, 6.43; Ala, 262; Val, 1.66; Ile, 0.79; Leu, 0.91; Lys,4.15 (24 hr hydrolysis in 4 N methane sulfonic acid, 110°).

I claim: 1.Ac-Ser(R¹)-Asp(OR²)-Ala-Ala-Val-Asp(OR²)-Thr(R³)-Ser(R¹)-Ser(R¹)-Glu(OR⁴)-Ile-Thr(R³)-Thr(R³)-Lys(R⁵)-Asp(OR²)-Leu-Lys(R⁵)-Glu(OR⁴)-Lys(R⁵)-Lys(R⁵)-Glu(OR.sup.4)-Val-Val-Glu(OR⁴)-Glu(OR⁴)-Ala-Glu(OR⁴)-Asn-OR⁶wherein Ac is acetyl; R¹ is a conventional protecting group for thehydroxyl group of the serine residue; R² is a conventional carboxylprotecting group; R³ is a conventional protecting group for the hydroxylgroup of threonine; R⁴ is a conventional carboxyl protecting group; R⁵is a conventional ω-amino protecting group; and R⁶ is a conventionalcarboxyl protecting group.
 2. The compound of claim 1 wherein R¹ isbenzyl, R² is benzyl, R³ is benzyl, R⁴ is benzyl, R⁵ isbenzyloxycarbonyl and R⁶ is benzyl. 3.Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys andpharmaceutically acceptable salts thereof. 4.Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn andpharmaceutically acceptable salts thereof. 5.Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn and pharmaceuticallyacceptable salts thereof.
 6. Glu-Val-Val-Glu-Glu-Ala-Glu-Asn andpharmaceutically acceptable salts thereof. 7.H-Asp(OBzl)-Leu-Lys(Z)-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzland the trifluoroacetic acid salt thereof, wherein Bzl is benzyl and Zis benzyloxycarbonyl. 8.Ac-Ser(Bzl)-Asp(OBzl)-Ala-Ala-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu-(OBzl)-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OHwherein Ac is acetyl, Bzl is benzyl and Z is benzyloxycarbonyl. 9.Ac-Ser(R¹)-Asp(OR²)-Ala-Ala-Val(OR²)-Asp(OR²)-Thr(R³)-Ser(R¹)-Ser(R¹)-Glu(OR⁴)-Ile-Thr(R³)-Thr(R³)-Lys(R⁵)-Asp(OR²)-Leu-Lys(R⁵)-Glu(OR⁴)-Lys(R⁵)-Lys(R⁵)-Glu(OR⁴)-Val-Val-Glu(OR⁴)-Glu(OR⁴)-Ala-Glu(OR.sup.4)-Asp-(CONH-CH(C₆H₅)- ##STR2## Resin)-OR² wherein Ac is acetyl; R¹ is a conventionalprotecting group for the hydroxyl group of the serine residue; R² is aconventional carboxyl protecting group; R³ is a conventional protectinggroup for the hydroxyl group of threonine; R⁴ is a conventional carboxylprotecting group; R⁵ is a conventional ω-amino protecting group and R⁶is a conventional carboxyl protecting group.
 10. The compound of claim 9wherein R¹ is benzyl, R² is benzyl, R³ is benzyl, R⁴ is benzyl, R⁵ is2,4-dichlorobenzyloxycarbonyl and R⁶ is benzyl.